Name: In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines (Video) | JoVE Journal
Uploaded: 2019-03-05T12:30:00.000+00:00
Duration: 9 min 57 s
Description: Learn in vitro assays to evaluate migration, invasion, and proliferation of immortalized human first-trimester trophoblast cell lines. These methods provide visual and quantitative analysis of factors influencing trophoblast invasion.

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5 minutes

The authors thank Dr. Gil Mor for providing the Sw.71 cells. This research was supported by an Albert McKern Scholar Award to S.W.

1. Cell Preparation
<ol>
	<li>Maintain cells in T-75 flasks in standard growth media at 37 &#176;C, 5% CO2, and 95% humidity. 
	NOTE: The cells used in these experiments were the immortalized first-trimester trophoblast cell lines Swan.71 (Sw.71) and HTR-8/SVneo9,10. The Sw.71 cells were maintained in DMEM/F-12 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1.0 mM sodium pyruvate, 10 mM HEPES, 0.10 mM MEM non-essential amino acids, 100 units/mL penicillin, and 100 &#181;g/mL streptomycin. The HTR-8/SVneo cells were maintained in RPMI-1640 supplemented with 5% heat-inactivated FBS.</li>
	<li>Allow the cells to replicate until they reach 90-100% confluence. Using a sterile tissue-culture flow hood, aspirate the media from the flask and wash cells with 10 mL of sterile 1x phosphate-buffered saline (PBS).</li>
	<li>Aspirate the PBS and add 5 mL of 1x (0.05%) trypsin-EDTA to the flask, making sure that cells are covered by trypsin. Place flask in the laboratory incubator maintained at 37 &#176;C, 5% CO2, and 95% humidity until all cells have detached from the bottom of the flask (approximately 5-10 min).</li>
	<li>Add 10 mL of pre-warmed growth media to the flask to deactivate the trypsin-EDTA.</li>
</ol>
2. Scratch Assay
<ol>
	<li>Following step 1.4, seed the appropriate number of cells into a 24-well plate to achieve 100% confluence in 48 h. 
	NOTE: It is possible to perform the scratch assay with a variety of different plate layouts. The maximum number of wells allowed by the wound maker tool is a 96-well format.</li>
	<li>The next day (day prior to scratch assay), change media to phenol-red free media supplemented as required, including heat-inactivated, charcoal dextran-stripped FBS. 
	NOTE: Other studies have recommended the use of media with low concentration FBS (e.g., 1%) to minimize cell proliferation, which can be used as an alternative in this protocol11.</li>
	<li>On the day of the scratch assay, pretreat cells for 30 min by adding desired treatment to the media in each well. The outlined experiments utilized vehicle (1x PBS) and 100 nM dexamethasone diluted in 1x PBS.</li>
	<li>To create uniform wounds, place sterile 10 &#181;L pipette tips on the pins of the wound maker tool. Lower the tips into the first row of wells and move the plate along the track of the wound maker until the pipette tips reach the other side of the well.
	<ol>
		<li>Move the plate back to the starting position, allowing the pipette tips to continuously touch the bottom of the plate to ensure a constant wound across the diameter of the well. Remove and replace the sterile 10 &#181;L pipette tips and lower the tips into the next row of wells.</li>
	</ol>
	</li>
	<li>Repeat step 2.4 until all wells are scratched. 
	NOTE: If a wound maker tool is not available, wounds can be created by manually scraping the center of the wells with a 10 &#181;L pipette tip.</li>
	<li>Carefully aspirate media and gently wash cells twice with 1x PBS. Following a final wash, add supplemented phenol-red free, stripped or low-FBS media with desired treatment or vehicle (as directed in step 2.3). 
	NOTE: HTR-8/SVneo cells should be washed with supplemented phenol-red media, as washing with 1x PBS will cause cells to detach.</li>
	<li>Place the 24-well plate containing scratched wells in the automated, time-lapse imager (see Table of Materials) housed in a laboratory incubator maintained at 37 &#176;C, 5% CO2, and 95% humidity. Image wells at regular intervals as needed to allow cells to complete wound healing (e.g., every 2 h for 72 h).</li>
	<li>Calculate the wound density and width by the software associated with the automated, time-lapse imager (see Table of Materials). To calculate the percentage change in wound size relative to the size of the starting wound, set the wound width at time 0 equal to 100%. Divide the wound size in each subsequent timepoint by the wound size at time 0 and multiply by 100 to obtain the percentage (Figure 1).</li>
</ol>
3. Invasion Assay
<ol>
	<li>Following step 1.4, seed the appropriate number of cells into 6-well plates to allow cells to reach 90% confluence in 48 h. Maintain cells in a laboratory incubator set to 37 &#176;C, 5% CO2, and 95% humidity.</li>
	<li>The next day, change media to phenol-red free media supplemented as required, including heat-inactivated, charcoal dextran-stripped FBS. 
	NOTE: Cells can be pretreated at this time depending on experimental design.</li>
	<li>At this time, place the vial of the 10 mg/mL extracellular matrix (see Table of Materials) on ice, and let it thaw in the refrigerator overnight.</li>
	<li>The next day, pre-cool invasion assay supplies by placing 24-well plates, cell culture inserts, microcentrifuge tubes, and pipette tips in the refrigerator (4 &#176;C) for at least 2 h prior to working with the extracellular matrix.</li>
	<li>Using chilled supplies in the tissue culture hood, dilute 10 mg/mL extracellular matrix 1:1 with phenol-red-free, serum-free media to a final concentration of 5 mg/mL. 
	NOTE: Always keep the stock and diluted extracellular matrix on ice. The ratio of extracellular matrix to media can be adjusted based on the properties of the cell line used in the assay.</li>
	<li>Using chilled supplies in the tissue culture hood, add 100 &#956;L of diluted extracellular matrix to the inserts (see Table of Materials) that have been placed in the 24-well plate. Make sure that the matrix is level without any bubbles. Place the 24-well plate containing matrix-coated inserts in the incubator at 37 &#176;C to allow the matrix to harden.</li>
	<li>To prep cells for invasion assay, wash cells with 1 mL of 1x PBS, aspirate the PBS, and add 500 &#181;L of 1x trypsin-EDTA to each well. Place the 6-well plate in the laboratory incubator maintained at 37 &#176;C, 5% CO2, and 95% humidity until all cells have detached from the bottom of the plate.</li>
	<li>Once cells have detached, add 500 &#181;L of phenol-red-free, serum-free media to each well of the trypsinized cells.</li>
	<li>Transfer 1,000 &#181;L of detached cells to microcentrifuge tube and spin down for 5 min at 400 x g at 4 &#176;C.</li>
	<li>Aspirate media and resuspend cells in phenol-red-free, serum-free media (volume will depend on concentration of cells). Count cells using an automated cell counter (see Table of Materials) and adjust volume of cell suspension for a final concentration of 5 x 105 cells per mL. 
	NOTE: A hemocytometer and microscope may also be used to count cells.</li>
	<li>Add 600 &#956;L of complete growth media to the wells of the 24-well plate that will be used for the invasion assay. 
	NOTE: Additional chemoattractants or treatments may be added to the complete growth medium in the well (lower chamber). The outlined experiments added vehicle (1x PBS) or 100 nM dexamethasone diluted in 1x PBS to the lower chamber.</li>
	<li>Place the pre-coated insert into each well containing complete growth medium that will be used for the invasion assay. Then add 200 &#956;L of cells on top of each pre-coated cell insert (upper chamber) for a total of 1 x 105 cells. 
	NOTE: As a negative control for this assay, an equal volume of phenol-red-free, serum-free media (without cells) can be added to the upper chamber.</li>
	<li>Place 24-well plate in the laboratory incubator maintained at 37 &#176;C, 5% CO2, and 95% humidity for 24 h. 
	NOTE: The 24 h incubation period was optimized for the immortalized first-trimester trophoblast cells and further tests may be required to determine the required incubation period in other cell lines.</li>
	<li>Following overnight incubation, suction the remaining media from the upper and lower chamber without disturbing the extracellular matrix. Then, carefully remove the extracellular matrix and non-invaded cells from the upper portion of the insert with a cotton swab, swabbing as many times as necessary to remove extracellular matrix.</li>
	<li>Wash each insert 3x with 1x PBS, adding PBS to both the upper and lower chambers of the well. Aspirate PBS after each wash.</li>
	<li>Fix cells attached to the inserts by placing inserts into 100% ice-cold methanol for 30 min at 4 &#176;C. Wash inserts 3x with 1x PBS and remove PBS after the final wash. Stain the cells attached to the inserts for 10 min with 0.2% crystal violet in 20% ethanol.</li>
	<li>Rinse 3x with deionized water and aspirate deionized water after the final wash.</li>
	<li>Air-dry inserts for 1 h at room temperature. Using forceps and a razor blade, carefully cut the bottom membrane of the insert and mount on a glass slide with the bottom side facing up. Allow mounting media to dry at room temperature.</li>
	<li>Obtain at least 4 unique images of invaded cells per sample using a light microscope with a 20x objective. Count the total number of cells in each image and normalize the number of cells to control samples and plot data (Figure 2).</li>
</ol>
4. Proliferation Assay
<ol>
	<li>Following step 1.4, count trypsinized cells from the flask using an automated cell counter and seed into 6-well plates at a density of 2 x 105 cells per well in standard growth media.</li>
	<li>Place cells in a laboratory incubator maintained at 37 &#176;C, 5% CO2, and 95% humidity for 4 h or until cells have adhered.</li>
	<li>Change media to phenol-red free media supplemented as required, including heat-inactivated, charcoal dextran-stripped FBS and add desired treatment. The outlined experiments added vehicle (1x PBS) or 100 nM dexamethasone diluted in 1x PBS. Place the 6-well plates in a laboratory incubator maintained at 37 &#176;C, 5% CO2, and 95% humidity.</li>
	<li>Remove media and replace with new media and treatment every 48 h.</li>
	<li>After 24, 48, or 72 h treatment, wash cells with 1 mL of 1x PBS and add 500 &#181;L of trypsin-EDTA to each well. Place 6-well plate in incubator until cells have detached from plate.</li>
	<li>Once cells have detached from the plate, add 500 &#181;L of the supplemented phenol-red free media to deactivate trypsin.</li>
	<li>Add 5 &#181;L of trypsinized cells to 5 &#181;L of Trypan blue in a separate tube and mix by pipetting.</li>
	<li>Count cells from each well in duplicate using an automated cell counter.</li>
	<li>Average the number of cells per well at each time point and plot as a function of time (Figure 3).</li>
</ol>

<ol><li>Ji, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Placental+trophoblast+cell+differentiation%3A+Physiological+regulation+and+pathological+relevance+to+preeclampsia%5BTitle%5D)+AND+%22Molecular+Aspects+of+Medicine%22%5BJournal%5D)">Placental trophoblast cell differentiation: Physiological regulation and pathological relevance to preeclampsia.</a> Molecular Aspects of Medicine. 34 (5), 981-1023 (2013).</li>
<li>Caniggia, I., Winter, J., Lye, S., Post, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxygen+and+placental+development+during+the+first+trimester%3A+implications+for+the+pathophysiology+of+pre-eclampsia%5BTitle%5D)+AND+%22Placenta%22%5BJournal%5D)">Oxygen and placental development during the first trimester: implications for the pathophysiology of pre-eclampsia.</a> Placenta. 21, S25-S30 (2000).</li>
<li>Norwitz, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defective+implantation+and+placentation%3A+laying+the+blueprint+for+pregnancy+complications%5BTitle%5D)+AND+%22Reproductive+Biomedicine+Online%22%5BJournal%5D)">Defective implantation and placentation: laying the blueprint for pregnancy complications.</a> Reproductive Biomedicine Online. 13 (4), 591-599 (2006).</li>
<li>Ness, R. B., Sibai, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shared+and+disparate+components+of+the+pathophysiologies+of+fetal+growth+restriction+and+preeclampsia%5BTitle%5D)+AND+%22American+Journal+of+Obstetrics+%26+Gynecology%22%5BJournal%5D)">Shared and disparate components of the pathophysiologies of fetal growth restriction and preeclampsia.</a> American Journal of Obstetrics & Gynecology. 195 (1), 40-49 (2006).</li>
<li>Knöfler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Critical+growth+factors+and+signalling+pathways+controlling+human+trophoblast+invasion%5BTitle%5D)+AND+%22The+International+Journal+of+Developmental+Biology%22%5BJournal%5D)">Critical growth factors and signalling pathways controlling human trophoblast invasion.</a> The International Journal of Developmental Biology. 54 (2-3), 269-280 (2010).</li>
<li>Huber, A. V., Saleh, L., Bauer, S., Husslein, P., Knöfler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TNF%CE%B1-Mediated+Induction+of+PAI-1+Restricts+Invasion+of+HTR-8%2FSVneo+Trophoblast+Cells%5BTitle%5D)+AND+%22Placenta%22%5BJournal%5D)">TNFα-Mediated Induction of PAI-1 Restricts Invasion of HTR-8/SVneo Trophoblast Cells.</a> Placenta. 27 (2), 127-136 (2006).</li>
<li>Nadeem, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nodal+Signals+through+Activin+Receptor-Like+Kinase+7+to+Inhibit+Trophoblast+Migration+and+Invasion%5BTitle%5D)+AND+%22The+American+Journal+of+Pathology%22%5BJournal%5D)">Nodal Signals through Activin Receptor-Like Kinase 7 to Inhibit Trophoblast Migration and Invasion.</a> The American Journal of Pathology. 178 (3), 1177-1189 (2011).</li>
<li>Onogi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypoxia+inhibits+invasion+of+extravillous+trophoblast+cells+through+reduction+of+matrix+metalloproteinase+%28MMP%29-2+activation+in+the+early+first+trimester+of+human+pregnancy%5BTitle%5D)+AND+%22Placenta%22%5BJournal%5D)">Hypoxia inhibits invasion of extravillous trophoblast cells through reduction of matrix metalloproteinase (MMP)-2 activation in the early first trimester of human pregnancy.</a> Placenta. 32 (9), 665-670 (2011).</li>
<li>Straszewski-Chavez, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+isolation+and+characterization+of+a+novel+telomerase+immortalized+first+trimester+trophoblast+cell+line%2C+Swan+71%5BTitle%5D)+AND+%22Placenta%22%5BJournal%5D)">The isolation and characterization of a novel telomerase immortalized first trimester trophoblast cell line, Swan 71.</a> Placenta. 30 (11), 939-948 (2009).</li>
<li>Graham, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+and+characterization+of+first+trimester+human+trophoblast+cells+with+extended+lifespan%5BTitle%5D)+AND+%22Experimental+Cell+Research%22%5BJournal%5D)">Establishment and characterization of first trimester human trophoblast cells with extended lifespan.</a> Experimental Cell Research. 206 (2), 204-211 (1993).</li>
<li>Liang, C. C., Park, A. Y., Guan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+scratch+assay%3A+a+convenient+and+inexpensive+method+for+analysis+of+cell+migration+in+vitro%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro.</a> Nature Protocols. 2 (2), 329-333 (2007).</li>
<li>Kisanga, E. P., Tang, Z., Guller, S., Whirledge, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucocorticoid+signaling+regulates+cell+invasion+and+migration+in+the+human+first-trimester+trophoblast+cell+line+Sw.+71%5BTitle%5D)+AND+%22American+Journal+of+Reproductive+Immunology%22%5BJournal%5D)">Glucocorticoid signaling regulates cell invasion and migration in the human first-trimester trophoblast cell line Sw. 71.</a> American Journal of Reproductive Immunology. , e12974(2018).</li>
<li>Berthois, Y., Katzenellenbogen, J. A., Katzenellenbogen, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phenol+red+in+tissue+culture+media+is+a+weak+estrogen%3A+implications+concerning+the+study+of+estrogen-responsive+cells+in+culture%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture.</a> Proceedings of the National Academy of Sciences of the United States of America. 83 (8), 2496-2500 (1986).</li>
<li>Cummings, B. S., Schnellmann, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+cell+death+in+mammalian+cells%5BTitle%5D)+AND+%22Current+Protocols+in+Pharmacology%22%5BJournal%5D)">Measurement of cell death in mammalian cells.</a> Current Protocols in Pharmacology. , Chapter 12, Unit 12 18 (2004).</li>
</ol>

The authors have nothing to disclose.

This procedure builds on the use of migration and invasion assays to include the rate of cell proliferation as a potential contributor to measured differences in trophoblast cell movement. Used together, these in vitro assays are simple and effective methods that can be used to identify the cellular pathways that regulate trophoblast migration. As described above, trophoblast cells may be treated with hormones, cytokines, growth factors, or other molecules to determine their impact on trophoblast movement. Alternatively,...

Placental development is a crucial step in the establishment of pregnancy, impacting both maternal and fetal health. However, the mechanistic basis by which this process occurs is not fully understood. Cell migration is an important biological process that contributes to the establishment and functions of the placenta during pregnancy. Following blastocyst implantation, the trophectoderm differentiates into villous trophoblasts, which cover the surface of the chorionic villi and are involved in gas and nutrient exchange, and extravillous trophoblasts (EVTs), which migrate out from the villi and invade the maternal decidua and vasculature1. Migration of the EVTs is essential for remodeling maternal spiral arteries and establishing uteroplacental circulation to support fetal growth2. Inadequate trophoblast invasion during pregnancy results in abnormal placental development and may contribute to pregnancy complications such as preeclampsia, gestational hypertension, intra-uterine growth restriction, and preterm birth3,4. Therefore, understanding the factors that affect trophoblast motility is essential to determining the pathways required for normal placentation.
Trophoblast migration is controlled by a complex network of signaling molecules, including growth factors, cytokines, hormones, and angiogenic factors5. Due to the limitations of studying the placenta in vivo, in vitro assays utilizing immortalized human trophoblast cell lines have been crucial to identifying factors that contribute to trophoblast motility. Scratch and invasion assays have been used extensively to quantitatively assess the role of individual molecules on trophoblast cell migration6,7,8. However, while useful in tandem to investigate how changes in migration may be caused by changes in cell invasiveness, these two assays do not account for additional mechanisms that may contribute to altered rates of cell migration. For example, reductions to the rate of cell proliferation may result in fewer cells available to migrate.
Here, we describe quantitative in vitro methods for assessing trophoblast migration using scratch, cell proliferation, and cell invasion assays. In the scratch assay, a uniform wound is created in a cell monolayer, and the migration of cells to fill the gap is measured by automated, time-lapse imaging of the wound size and density of cells within the wound. The cell proliferation assay is based on calculating the ratio of cells at each time point compared to a known starting quantity of cells. In the cell invasion assay, cells are seeded atop an extracellular matrix-coated cell culture insert chamber (e.g. Transwell), and the number of cells that invade through the extracellular matrix in response to chemo-attractant are counted.
The scratch assay is a simple and effective tool that may be used to determine how different environmental conditions affect cell migration. The proliferation and invasion assays may subsequently be used to determine the contribution of cell proliferation and invasiveness to overall changes in cell migration. Collectively, these assays provide robust measurements of biological processes that may contribute to cell motility. Two immortalized first-trimester trophoblast cell lines were used in the described assays, Swan.71 (Sw.71) and HTR-8/SVneo9,10. However, these assays may also be optimized for use in other cell types to identify key modulators of cell migration and invasion.

<table><tbody><tr><td>0.4% Trypan blue stain</td><td>Thermo Fisher Scientific</td><td>15250-061</td><td></td></tr><tr><td>0.5% Trypsin-EDTA</td><td>Life Technologies</td><td>15400-054</td><td></td></tr><tr><td>1.0 M HEPES</td><td>AmericanBio</td><td>AB06021-00100</td><td></td></tr><tr><td>10 mM MEM non-essential amino acids</td><td>Life Technologies</td><td>11140-050</td><td></td></tr><tr><td>100 mM sodium pyruvate</td><td>Life Technologies</td><td>11360-070</td><td></td></tr><tr><td>24-well plate transwell inserts</td><td>Corning</td><td>3422</td><td>Contain polycarbonate filters with an 8 &amp;mu;m pore size</td></tr><tr><td>Charcoal dextran-stripped FBS</td><td>Gemini Bio-Products</td><td>100-119</td><td></td></tr><tr><td>Countess II Automated Cell Counter</td><td>Thermo Fisher Scientific</td><td>AMQAX1000</td><td></td></tr><tr><td>Crystal Violet</td><td>Sigma Aldrich</td><td>C0775</td><td></td></tr><tr><td>Cytoseal 60</td><td>Thermo Fisher Scientific</td><td>8310-4</td><td></td></tr><tr><td>Dexamethasone (Dex; 1, 4-pregnadien-9&amp;alpha;-fluoro-16&amp;alpha;-methyl-11&amp;beta;, 17, 21-triol-3, 20-dione; &amp;ge;98% TLC)</td><td>Steraloids</td><td>P0500-000</td><td></td></tr><tr><td>DMEM/Ham&amp;rsquo;s F12</td><td>Life Technologies</td><td>11330-032</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma Aldrich</td><td>F2442</td><td></td></tr><tr><td>IncuCyte 24-well ImageLock Plates</td><td>Essen BioScience</td><td>4365</td><td>If using the IncuCyte Zoom imaging system, seed cells onto IncuCyte ImageLock Plates. These plates have fiducial markers on the bottom of the plate that are used as a reference for repeat imaging in a constant field of view.</td></tr><tr><td>IncuCyte software</td><td>Essen BioScience</td><td>2010A Rev2</td><td></td></tr><tr><td>IncuCyte ZOOM system</td><td>Essen BioScience</td><td>N/A</td><td>Scratch assay images were captured and analyzed using the preset &amp;ldquo;Scratch Wound&amp;rdquo; parameters on the IncuCyte ZOOM system. Other time-lapse microscopes may also be used.</td></tr><tr><td>Matrigel Membrane Matrix</td><td>Becton Dickinson</td><td>356231</td><td>Growth factor reduced, phenol-red free</td></tr><tr><td>Micro Slides</td><td>VWR International, LLC</td><td>48311-7003</td><td></td></tr><tr><td>Microscope cover glass</td><td>Fisher Scientific</td><td>12-545-E</td><td></td></tr><tr><td>Olympus IX71 inverted microscope</td><td>Olympus</td><td>IX71</td><td></td></tr><tr><td>Penicillin (10,000 units/mL)/streptomycin (10,000 &amp;micro;g/mL)</td><td>Life Technologies</td><td>151-40-122</td><td></td></tr><tr><td>Phenol-red free DMEM/Ham&#039;s F12</td><td>Life Technologies</td><td>11039-021</td><td></td></tr><tr><td>Semi-automatic wound maker tool</td><td>Essen BioScience</td><td>N/A</td><td></td></tr></tbody></table>

in vitro assays to evaluate the migration, invasion, and proliferation of immortalized human first-trimester trophoblast cell lines

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

The immortalized human first-trimester trophoblast cell lines Sw.71 and HTR-8/SVneo were used in these experiments to determine the role of glucocorticoids in trophoblast cell migration12. Glucocorticoids were used in these experiments as they have recently been shown to alter trophoblast functions, although alternative treatments could be used12. Both cell lines were treated with vehicle (1x PBS) or 100 nM of the synthetic glucocorticoid de...

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...

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A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

The present protocol allows the recovery of tube-like structures formed in the classical in vitro angiogenesis-tube formation assay where cells, such as trophoblast cells, organize in capillary-like structures over a layer of extracellular basement membrane matrix. The recovered structures are used for RNA and protein isolation for various biochemical analyses.

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

This protocol enables purification, characterization and culture of primary HLA-G+ extravillous trophoblasts (EVT) from the decidua basalis and chorionic membrane of human term placentas. Primary EVT cultures maintain viability for 96 h, allowing co-culture with sample matched maternal immune cells and subsequent flow cytometric and molecular analysis of their interactions.

Purification of HLA-G+ Extravillous Trophoblasts from Human Term Placental Tissues for Phenotyping and Functional Analysis

Learn in vitro assays to evaluate migration, invasion, and proliferation of immortalized human first-trimester trophoblast cell lines. These methods provide visual and quantitative analysis of factors influencing trophoblast invasion.

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines (Video) | JoVE Journal

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

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